It is fairly well established that the cholesterol content of whole blood is directly related to certain ailments in humans and animals. Among these ailments are hepatocellular diseases, thyroid metabolism diseases, biliary obstruction, and atherosclerosis and other vascular difficulties. Cholesterol is found in blood in either its free form or in the form of its ester. Free cholesterol refers to cholesterol in its unreacted state. Total cholesterol refers to the sum of free cholesterol and its ester derivatives. Cholesterol is found in constant amounts in serum under normal conditions. In general, 25% of the total cholesterol level in serum is free cholesterol while the remaining 75% is in the form of ester derivatives.
Known quantitative analyses of total cholesterol involve the use of enzymes to convert both free cholesterol and cholesterol esters into detectable products. This enzymatic method uses the following series of reactions involving cholesterol esterase, cholesterol oxidase and peroxidase. ##STR1##
In the first reaction, it is essential that all of the cholesterol esters are hydrolyzed to free cholesterol. This can be accomplished with a primary hydrolyzing reagent which is an enzyme having cholesterol esterase activity. This enzyme is known in the art as lipase, esterase or cholesterol ester hydrolase. However, it has been reported by Wiebe et al in Clin. Chem., 30(3), pp. 352-356 (1984) and references cited therein that incomplete hydrolysis can occur thereby giving inaccurately low cholesterol determinations. Many attempts have been made to augment ester hydrolysis by adding biliary cofactors, protease or nonionic surfactants (e.g. the TRITONs available from Rohm & Haas, Philadelphia, Pa.).
A recent advance in the clinical chemistry art is a multilayer analytical element for the determination of total cholesterol manufactured by Eastman Kodak Company (Rochester, N.Y.) under the label of EKTACHEM Clinical Chemistry Slide (CHOL) and described in Kodak Publication MP2-35, 1983 entitled "Kodak EKTACHEM Clinical Chemistry Products Test Methodology for Cholesterol Analysis." This element contains reagents which hydrolyze cholesterol esters and determine total cholesterol in the test sample. For example, it contains about 540 I.U./m.sup.2 of cholesterol ester hydrolase and about 11 g/m.sup.2 of a nonionic surfactant.
It has been observed, however, that the described element is sensitive to fluctuating levels of triglycerides present in the serum sample. For example, elevated triglyceride levels can positively bias the total cholesterol determination. User attempts to minimize this effect involve dilution procedures which are time-consuming and subject to additional error.
Merely adding increased amounts of a single reagent to the element does not eliminate the adverse effect of the triglycerides because of the multiple reactions used in the assay. Hence, there is a need in the art for a means for determining total cholesterol in a liquid sample which is not adversely affected by the amount of triglycerides in the sample.